Coordinate regulation of virulence determinants in the agent of human cholera, Vibrio cholerae, is directed by the ToxR protein. ToxR is proposed to act by controlling expression of another regulator protein, encoded by the toxT gene, which activates genes required for virulence. The goals of this research proposal are to determine how ToxR controls toxT expression and to understand the role of ToxT in this regulatory cascade. The nucleotide sequence of toxT will be determined and its product will be characterized by biochemical and immunochemical means. The mechanism of ToxR control over toxT expression will be defined by identifying and analyzing the toxT transcript and its promoter using genetic and biochemical methods. A toxT mutant strain of V. cholerae will be constructed and characterized in vitro and in animal models. Two dimensional gel and reporter gene fusion analyses will be used to determine if there are proteins regulated by ToxT that are independent of ToxR, and vice versa. The promoter of a ToxT activated gene, tagA, will be identified and characterized by mutation analysis and DNA binding experiments. The role of ToxT in temperature control of virulence genes will be determined by a combination of in vitro and in vivo experiments. Recent evidence suggests that regulatory cascades may act in virulence gene expression in other bacteria, although components of the cascades in these systems have yet to be identified. Thus, the ToxR-ToxT system in V. cholerae, represents a model for how a regulatory circuit operates in pathogenesis. In addition, knowledge obtained by studying regulation in V. cholerae is essential to the development of good live, oral vaccines against cholera.